Food safety and prevention of food spoilage is an ever present concern worldwide, particularly within the meat industry. Spoilage of food is a major economic problem for a food manufacturer. Food manufacturers need to protect the health and safety of the public by delivering products that are safe to eat. Such food must have a guaranteed shelf life, either at chilled or ambient temperature storage. Consumers prefer good tasting food of high quality. This is difficult to achieve with chemical preservatives, harsh heating regimes and other processing measures. Food safety and protection is best achieved with a multiple preservation system using a combined approach of milder processing and natural preservatives. Foodborne micro-organisms are also less able to adapt and grow in food preserved with different preservative measures.
There is much concern about food protection and the growth of food spoilage organisms such as Listeria monocytogenes. This particular species is one of the most problematic spoilage microorganism in meat. The unusual physiological characteristics such as exceptional resistance toward antimicrobials are largely responsible for their ability to cause spoilage. Additionally, spoilage organisms can sometimes adapt to different preservatives and storage conditions, thus a combination of preservative measures can be more successful than individual measures.
There is an increasing need to develop economical, natural and effective preservative systems to meet the public demand for convenient, natural, safe, healthy, good quality products with guaranteed shelf life. Antimicrobial materials such as those derived from plants can be used as preservatives in food to help meet this need. Such plant extracts are considered to be desirable because they are regarded as being natural. Moreover from a regulatory point of view, because of long term usage, plant extracts typically have GRAS (generally regarded as safe) status. There is also a continuing to desire to provide microbial protection utilizing lower amounts of antimicrobial materials. Thus there is a need to provide new antimicrobial materials or new more effective combinations of antimicrobial materials.
Despite their natural origins, it is desirable that antimicrobial products from plants be used in the lowest possible amounts. This is desirable not only for reasons of cost but also to meet consumer desire to minimize the amount of ‘additives’ in foodstuffs. Moreover, many plant materials have an associated taste. Therefore in many demanding food applications reduction of the amount of protectant from plant origin is advantageous.
Meat manufacturers are looking for ways to enable them to supply retail outlets from efficient, cost effective, central-processing centers. Increased shelf life with regard to spoilage (consumer safety) is required to make this possible as meat makes its way through longer distribution channels from producer to retailer to consumer.
Color shelf life is important to consumer acceptance. Consumers judge the freshness of meat by the presence of bright red oxymyoglobin pigment. Oxymyoglobin in fresh meat decreases with time during storage as it changes to the stable brown pigment, metmyoglobin. Although oxymyoglobin pigment fades during dark storage, for example in a meat locker, pigment loss is most pronounced in lighted, refrigerated display cases in retail establishments. Further, pigment loss is primarily cosmetic in nature, it has serious economic consequences. Consumers in search of the freshest looking cuts avoid purchasing meat containing even small amounts of brown metmyoglobin.
Shelf life associated with microbial spoilage is a serious issue. The potential liability associated with food borne illness outbreaks from the sale of microbe-contaminated meat is enormous. The meat industry and associated retail outlets are seeking ways to insure consumers' safety by preventing microbial contamination all along the manufacturing process. Process improvements such as carcass washing and carefully controlled low temperature processing are now routine in the industry. One method for increasing shelf life associated with microbial spoilage is to package the food, e.g. meat, using modified atmosphere package (MAP).
There is a need in the industry for antimicrobial methods and processes which are perceived by consumers as being more natural. The antimicrobial activity of the composition comprising Lamiaceae extract and hesperidin has been the main subject of study. Most prior art indicate that the antimicrobial activity of the herbs is centered in the volatile essential oil components.
P. M. Davidson and A. S. Naidu (in Natural Food Antimicrobial Systems, A. S. Naidu, ed., 2000, CRC Press, Boca Raton, pages 265-294) review the antimicrobial properties of phyto-phenolic compounds from essential oils of spices, herbs, edible grains and seeds. The authors teach that the antimicrobial effects of spices and herbs are primarily due to the presence of phenolic compounds in the essential oil fractions and that some monoterpenes seem to show some activity, as well. Carvacrol, p-cymene and thymol are identified as the major volatile components of oregano, thyme and savory that likely account for the observed activity. The active antimicrobial agents of rosemary have been suggested to be borneol, camphor, 1,8-cineole, alpha pinene, camphene, verbenone and bornyl acetate. The active constituent of sage has been suggested to be thujone. Minimum lethal concentrations of essential oils of thyme oil have been shown to range from 225-900 ppm in cultures. These concentrations of essential oils in foods would cause serious flavor problems. Since culture experiments underestimate the concentration necessary for effectiveness in foods, the flavor problems in foods are likely to be more serious than even the culture numbers suggest. In another portion of this reference, minimum inhibitory concentrations of essential oils were stated as 1-2% for rosemary, 0.12-2% for thyme, 0.12-2% for spearmint, 0.5-2% for sage, 0.5-2% for peppermint and 0.12-2% for oregano. In the summary, the authors state that concentrations of antimicrobial compounds in herbs and spices are too low to be used effectively without adverse effects on the sensory characteristics of a food.
Y. Kimura et al. in U.S. Pat. No. 4,380,506, teach a process for producing a preservative having antioxidant and antimicrobial activity. The process involves partitioning an extract of herb spices between polar and non-polar solvents. Some of the partitioned extracts showed antimicrobial activity against Gram positive Bacillus subtilis microorganisms in culture media. The only taste criterion tested by Kimura et al. was the bitterness. Kimura et al. remained silent as to essential oil taste perception. Kimura et al. did not deodorize the extract which means that the extract contained essential oils and impacted the taste of the meat. This impact on taste teaches away from using rosemary extracts obtained by the process taught by Kimura et al.
D. Ninkov (WO 01/15680) teaches that pharmaceutical compositions can be prepared by combining extracts of essential oils from plants of the Lamiaceae family with an organic acid. Ninkov teaches that the antimicrobial activity of the pharmaceutical composition is due to the presence of organic phenols such as isopropyl o-cresol in the oil extract from the plant.
K. Shetty and R. G. Labbe, (Asia Pacific J. Clin. Nutr. (1998), 7(3/4), pages 270-276., describe work to clone Lamiacae plants to produce enhanced levels of essential oil components such as carvacrol and thymol. These essential oil components have some antimicrobial properties but their commercial use is prevented by the strong flavors imparted to foods by these volatile compounds.
J. Campo, M. Amiot and C. Nguyen-the (2000, Journal of Food Protection 63, pages 1359-1368) teach that rosemary extract has antimicrobial properties in culture studies. Minimum inhibitory concentrations varied with the species of bacteria being tested, but ranged from 0.06-1%. These researchers suggest that rosemary extract may show promise in foods with low fat and low protein content, against Gram positive organisms. No food systems were actually studied in this reference. This reference did not study specifically Listeria. 
E. Down, et al., “Comparison of Vitamin E, Natural Antioxidants and Antioxidant Combinations on the Lean Color and Retail Case-Life of Ground Beef Patties” published in October, 1999, describes the effect of rosemary extract in combination with other natural antioxidants and vitamin E diet supplementation on the color life of non-MAP ground beef. This reference does not teach how to extend the microbial shelf life of the meat. The authors failed to demonstrate a red color improvement of the meat by using rosemary as the red color preservation in meat with a natural antioxidant containing rosemary could not statistically differ from the control. The red color of the control alters within commercially desirable period. The loss of as much of the red color in the control as in meat with the rosemary from this reference teaches away from using rosemary extract as stability agent capable of preserving the red color of the meat.
Ahn et al. “Effects of plant extracts on microbial growth, color change, and lipid oxidation in cooked beef”, Food Microbiol., Vol. 24, Issue 1, (2007): 7-14 show that rosemary extract, or rosemary oleoresin, for which the contents in phenolic diterpenes are not known, has an antilisterial effect. In this reference, grape seed extract and pine bark extract had a greater antilisterial effect than rosemary which teaches away from using rosemary extract as the lead antilisterial natural product in meat. Further, Ahn et al. 2007 have shown that the addition of rosemary extract to meat significantly deteriorated the red color of the meat, as compared to the control that lost less of the red color or as compared to grape extract that significantly improved the preservation of the red color of the meat. Therefore, Ahn et al. 2007 teach one to not use rosemary extract as stability agent capable of preserving the red color of the meat.
United States Patent Application Publication No. 2004/131709 studies show that rosemary extract alone, Herbalox® Seasoning, in which the majority of the volatile oil components has been removed shows very little, if any, antimicrobial effect. This reference does not teach how to extend the Gram positive, more precisely antilisterial shelf life of meat.
In addition, plant derived antimicrobials from citrus reported in the prior art are acids not flavonoids. For example, prior patents directed to compounds from citrus essentially relate to acids. KR20040001441 describes orange juice as a suppressor of germ growth. However, only less than approximately 1/50th of the juice reported in KR20040001441 could be used in meat without perceiving meat as sour. As meat takes up only up to 7.2% of the solution rich in citric acid, final levels in hesperidin taken up in meat would then correspond to less than 0.48%* 1/50*7.2%=˜0.0007%. This reference does not teach whether hesperidin could have an antilisterial effect in meat.
Lorente, José et al. “Chemical guide parameters for Spanish lemon (Citrus limon (L.) Burm.) juices.” Food chemistry 162 (2014): 186-191 discloses that citrus juice has titratable acidity of 52.4 g/L, with citric acid being the main component. According to Lorente et al. (2014) in such juice, hesperidin levels as compared to titratable acidity are lower by more than two orders of magnitude (257 to 484.8 mg/L), which corresponds to 0.26 to 0.48% hesperidin w/v. Adding such an acidic composition to meat would impact the meat taste already at low levels.
Aktaş, Nesimi, and Mükerrem Kaya. “The influence of marinating with weak organic acids and salts on the intramuscular connective tissue and sensory properties of beef.” European Food Research and Technology 213.2 (2001): 88-94 show that adding a solution of from 1% weak acid (including citric acid) to meat confers to the meat the sour taste. Also they show that when marinated in proportions 1:1 w/v (meat/marinade) the meat gains in weight at most 7.2% following marinating in marinades containing citric acid.
In WO 2012/112337, it is reported that flavonoids, including hesperidin, may provide some active antimicrobial activity without informing on the nature of microbes, whether they are bacteria, whether they are Gram positive bacteria nor whether they are Listeria. WO 2012/112337 teaches that active antimicrobial compounds are acids.
Moulehi, Ikram, et al. “Variety and ripening impact on phenolic composition and antioxidant activity of mandarin (Citrus reticulate Blanco) and bitter orange (Citrus aurantium L.) seeds extracts.” Industrial Crops and Products 39 (2012): 74-80 report that citrus seed extracts contain total flavonoids of 1.31 to 2.52 mg equivalent catechins/g DW. As hesperidin represents <16% of total flavonoids of citrus seed extract, this means that hesperidin is present at ˜0.032% in DW citrus seed extract.
Mandalari, G., et al. “Antimicrobial activity of flavonoids extracted from bergamot (Citris bergamia Risso) peel, a byproduct of the essential oil industry.” Journal of Applied Microbiology 103.6 (2007): 2056-2064 disclose that in vitro, citrus extracts rich in flavonoids inhibit the growth of Gram negative bacteria only and have no effect on the growth of Gram positive bacteria, and have no effect on the growth of Listeria. Mandalari et al. (2007) show that neohesperidin in pure form has no effect on Listerial growth in vitro.
Fernandez-Lopez, J., et al. “Antioxidant and antibacterial activities of natural extracts: application in beef meatballs.” Meat science 69.3 (2005): 371-380 show that meat supplemented with citrus extracts containing flavonoids, the main of which is hesperidin, has no effect on the growth of Listeria monocytogenes. For example, Fernandez-Lopez et al. (2005) show that such extracts exert antimicrobial effects on other bacterial strains, including Listeria innocua, but not on Listeria monocytogenes. 
Teachings of Mandalari et al. and Fernandez-Lopez et al. teach away from using hesperidin as antilisterial compound and do not render obvious to use any or combination of flavonoids from a citrus extract against Listeria monocytogenes in meat. For example, Mandalari et al. (2007) teach away from using hesperidin as antilisterial compound and do not render obvious that a purified flavonoid could have an antilisterial effect.
Punica extracts rich in ellagic acid have no antimicrobial effects in raw MAP meat. For example, Hayes et al. (Hayes, J. E., Stepanyan, V., Allen, P., O'Grady, M. N., & Kerry, J. p. 2010). “Effect of lutein, sesamol, ellagic acid and olive leaf extract on the quality and shelf-life stability of packaged raw minced beef patties”, Meat science, 84(4), 613-620.) (hereinafter “Hayes et al”) teach that ellagic acid (one of active compounds from the Punica extract) has no antimicrobial effect on raw beef MAP meat stored in cold and when applied at 300 ppm. Hayes et al. teach that ellagic acid did not improve the preservation of the red color of raw beef MAP meat stored in cold and when applied at 300 ppm or at 600 ppm. Hayes et al. teach away from using lower concentrations than 300 ppm in ellagic acid for antimicrobial effect. Hayes et al. teach away from using ellagic acid for improving the red color of meat.
The general problem of enhancing the shelf life of fresh meat without impacting the taste, remains in preventing the growth of spoilage organisms and pathogens and in preserving the red color of the meat throughout the commercially desirable storage period.